Growth of tumors and metastasis are processes known to require neovascularization. To ascertain the participation of endogenous angiogenesis inhibitors in tumor progression and as part of our previous grant cycle, we generated tumor-prone mice (by virtue of c-neu overexpression in the mammary gland) that either over expressed or lack thrombospondin-l (TSP1). In TSP1 deficient animals, tumor burden and vasculature were significantly increased, tumor capillaries were distended, sinusoidal in shape and showed greater fragility. In contrast, TSP1 over expressors showed delayed tumor growth or lacked frank tumor development (20% of animals), tumor vessels were thin and less frequent. Together these findings argue for a protective role of endogenous angiogenesis inhibitors in tumor progression and they also revealed the contribution of TSP1 to vascular morphogenesis. In order to dissect the molecular mechanisms behind these effects we evaluated the levels and status of a large number of extracellular matrix molecules and growth factors. Interestingly, absence of TSP1 was associated with higher levels of active matrix metalloproteinase-9 (MMP9), a molecule shown to facilitate both angiogenesis and tumor invasion. In contrast, tumors with excess of TSP1 showed a marked reduction in the overall levels of active MMP9. In vitro, enzymatic activation of pro-MMP9 by two endogenous activators (MMP3 and p!asmin) was suppressed by TSP1 in a dose-dependent manner. These findings lead us to the hypothesis that TSP1 is a regulator of MMP9 processing/activation, and many of its effects, in particular those related to angiogenesis might rely on this function. The present competitive renewal grant application aims to further dissect the molecular regulation of MMP9 activation by TSP1 and to explore the relevance of these findings using a combination of in vitro biochemical approaches and comprehensive animal models. We anticipate that information gathered from the execution of the experiments proposed will increase our current understanding of the mechanisms of TSP1 function by placing this molecule 'in context" of proteases and growth factors, all participant of the tumor angiogenesis cascade.